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1.
Genome Res ; 23(9): 1446-61, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23796952

ABSTRACT

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.


Subject(s)
Chromosomal Instability , Chromosomes, Human, Pair 8/genetics , Colonic Neoplasms/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Case-Control Studies , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasm Metastasis/genetics , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factor 7-Like 1 Protein/genetics , Transcription Factor 7-Like 1 Protein/metabolism , Transcription, Genetic , Wnt Signaling Pathway
2.
JAMA ; 305(1): 59-67, 2011 Jan 05.
Article in English | MEDLINE | ID: mdl-21205967

ABSTRACT

CONTEXT: Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. OBJECTIVE: To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. DESIGN, SETTING, AND PATIENTS: CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. MAIN OUTCOME MEASURES: Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. RESULTS: In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). CONCLUSIONS: A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.


Subject(s)
Chromosome Deletion , Genes, p53/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , MicroRNAs/genetics , Adult , Aged , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 17/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Prognosis , Transcription, Genetic , Tumor Suppressor Protein p53/physiology , ZAP-70 Protein-Tyrosine Kinase/physiology
3.
Blood ; 116(6): 945-52, 2010 Aug 12.
Article in English | MEDLINE | ID: mdl-20393129

ABSTRACT

Aberrant expression of microRNAs (miRNAs) has been associated with clinical outcome in patients with chronic lymphocytic leukemia (CLL). To identify a powerful and easily assessable miRNA bio-marker of prognosis and survival, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) profiling in 104 CLL patients with a well-defined chromosome 17p status, and we validated our findings with miRNA microarray data from an independent cohort of 80 patients. We found that miR-15a, miR-21, miR-34a, miR-155, and miR-181b were differentially expressed between CLLs with chromosome 17p deletion and CLLs with normal 17p and normal karyotype, and that miR-181b was down-regulated in therapy-refractory cases. miR-21 expression levels were significantly higher in patients with poor prognosis and predicted overall survival (OS), and miR-181b expression levels significantly predicted treatment-free survival. We developed a 21FK score (miR-21 qRT-PCR, fluorescence in situ hybridization, Karyotype) to stratify patients according to OS and found that patients with a low score had a significantly longer OS time. When we evaluated the relative power of the 21FK score with the most used prognostic factors, the score was the most significant in both CLL cohorts. We conclude that the 21FK score represents a useful tool for distinguishing between good-prognosis and poor-prognosis CLL patients.


Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 17 , Genetic Testing/statistics & numerical data , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , MicroRNAs/genetics , Adult , Aged , Aged, 80 and over , Female , Genetic Markers , Genetic Testing/standards , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Karyotyping , Male , Middle Aged , Nucleotide Mapping , Predictive Value of Tests , Prognosis , Reproducibility of Results , Risk Factors
4.
PLoS One ; 4(10): e7405, 2009 Oct 12.
Article in English | MEDLINE | ID: mdl-19823581

ABSTRACT

BACKGROUND: The physiopathology of sepsis continues to be poorly understood, and despite recent advances in its management, sepsis is still a life-threatening condition with a poor outcome. If new diagnostic markers related to sepsis pathogenesis will be identified, new specific therapies might be developed and mortality reduced. Small regulatory non-coding RNAs, microRNAs (miRNAs), were recently linked to various diseases; the aim of our prospective study was to identify miRNAs that can differentiate patients with early-stage sepsis from healthy controls and to determine if miRNA levels correlate with the severity assessed by the Sequential Organ Failure Assessment (SOFA) score. METHODOLOGY/PRINCIPAL FINDINGS: By using genome-wide miRNA profiling by microarray in peripheral blood leukocytes, we found that miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiated sepsis patients from healthy controls. We also proved by quantitative reverse transcription-polymerase chain reaction that miR-150 levels were significantly reduced in plasma samples of sepsis patients and correlated with the level of disease severity measured by the SOFA score, but were independent of the white blood counts (WBC). We found that plasma levels of tumor necrosis factor alpha, interleukin-10, and interleukin-18, all genes with sequence complementarity to miR-150, were negatively correlated with the plasma levels of this miRNA. Furthermore, we identified that the plasma levels ratio for miR-150/interleukin-18 can be used for assessing the severity of the sepsis. CONCLUSIONS/SIGNIFICANCE: We propose that miR-150 levels in both leukocytes and plasma correlate with the aggressiveness of sepsis and can be used as a marker of early sepsis. Furthermore, we envision miR-150 restoration as a future therapeutic option in sepsis patients.


Subject(s)
MicroRNAs/blood , MicroRNAs/metabolism , Sepsis/blood , Sepsis/microbiology , Aged , Female , Humans , Interleukin-10/blood , Interleukin-18/blood , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Prognosis , Prospective Studies , Sepsis/mortality , Treatment Outcome , Tumor Necrosis Factor-alpha/blood
5.
Curr Pharm Des ; 14(21): 2040-50, 2008.
Article in English | MEDLINE | ID: mdl-18691114

ABSTRACT

MicroRNAs (miRNAs) are an abundant class of approximately 22-nucleotide-noncoding RNAs, which play important regulatory roles in animal and plant development: they are involved in gene expression at the posttranscriptional level by degrading or blocking translation of messenger RNA (mRNA) targets. miRNAs can induce RNA cleavage and chromatin modifications, and are implicated in apoptotic pathways and regulation of cell growth and proliferation. It is becoming clear that miRNAs play important roles in the regulation of gene expression during development, and our knowledge of the expression levels or function of miRNAs in normal and neoplastic cells is increasing. Accumulating experimental evidence suggests that different miRNAs are deregulated in primary human tumors and that many human miRNAs are located at genomic regions linked to cancer. miRNAs may be important regulators of mammalian hematopoiesis. They are involved in a variety of hematological malignancies, including acute lymphoblastic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and primary effusion lymphoma. Here, we provide background on the biogenesis and function of miRNAs and discuss potential therapeutic applications of miRNA-based technology in hematological malignancies.


Subject(s)
Gene Expression Regulation, Neoplastic , Hematologic Neoplasms/therapy , MicroRNAs/metabolism , Animals , Hematologic Neoplasms/genetics , Hematopoiesis/genetics , Humans , RNA, Messenger/metabolism
6.
Int J Cancer ; 122(5): 969-77, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-18098138

ABSTRACT

MicroRNAs (miRNAs) represent a new class of small noncoding RNAs (ncRNAs, RNAs that do not codify for proteins) that can regulate gene expression by targeting messenger RNAs of protein coding genes and other ncRNA transcripts. miRNAs were recently found to be involved in the pathophysiology of all types of analyzed human cancers mainly by aberrant gene expression. This is characterized by abnormal levels of expression for mature and/or precursor miRNA transcripts in comparison to the corresponding normal tissues. miRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of human tumors. Therefore, miRNAs fingerprinting represents a new addition to the tools to be used by medical oncologists.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , MicroRNAs , Neoplasms/genetics , Animals , Humans
7.
Clin Transl Sci ; 1(1): 50-6, 2008 May.
Article in English | MEDLINE | ID: mdl-20443818

ABSTRACT

Abstract MicroRNAs (miRNAs) are a large family of short, single-stranded, highly conserved noncoding RNAs involved in gene regulation that can regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs). miRNAs have been implicated in the development of a wide variety of cancers as well as heart disease and other diseases. This review describes the role of miRNAs in human disease, methodology for evaluating miRNA gene expression, and the potential role of miRNAs as therapeutic agents and targets for the treatment of disease.


Subject(s)
Gene Expression Regulation , Genomics/methods , Heart Diseases/metabolism , MicroRNAs/metabolism , Neoplasms/metabolism , Animals , Caenorhabditis elegans , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Models, Biological , Tissue Distribution
8.
J Cell Biochem ; 104(2): 595-605, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18092340

ABSTRACT

We have compared the cytotoxic/cytostatic responses of the SKW6.4 lymphoblastoid B-cells to the alkylating agent chlorambucil, the purine analog fludarabine, the non-genotoxic activator of the p53 pathway, Nutlin-3, used alone or in association with the death-inducing ligand recombinant TRAIL. Exposure to chlorambucil, fludarabine, and Nutlin-3 induced p53 accumulation and variably affected cell cycle progression in SKW6.4 lymphoblastoid cells. In particular, chlorambucil induced cell cycle accumulation at the G2/M checkpoint; Nutlin-3 induced early cell cycle arrest at the G1/S checkpoint, while fludarabine showed an intermediate behavior. On the other hand, recombinant TRAIL alone did not affect cell cycle progression but induced a rapid increase of apoptosis. Analysis of the gene expression profile of the p53-transcriptional targets showed distinct features between chlorambucil, Nutlin-3 and fludarabine, which likely account for their differential effect on cell cycle in SKW6.4 cells. In particular, chlorambucil upregulated the steady-state mRNA expression of SFN/14-3-3sigma, a gene involved in G2/M cell cycle arrest. Of note, all agonists upregulated TRAIL-R2 expression in SKW6.4 cells both at the mRNA and protein levels. Consistently, pretreatment with chlorambucil, fludarabine and Nutlin-3 enhanced SKW6.4 sensitivity to TRAIL-mediated apoptosis.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Cell Cycle/drug effects , Imidazoles/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , B-Lymphocytes/pathology , Blast Crisis/pathology , Cell Line, Tumor , Chlorambucil/pharmacology , Humans , Up-Regulation/genetics , Vidarabine/analogs & derivatives , Vidarabine/pharmacology
9.
J Cell Physiol ; 213(1): 229-36, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17476690

ABSTRACT

Among 14 peripheral blood samples obtained from patients affected by B chronic lymphocytic leukemia (B-CLL) at initial stages (Rai 0-1) of the disease, 6 showed intermediate/high levels of Zap-70 while 8 displayed low/absent levels of Zap-70. Although Zap-70(high) and Zap-70(low) B-CLL samples displayed similar levels of surface death receptor TRAIL-R2, recombinant TRAIL induced cytotoxicity only in a subset of Zap-70(low) B-CLL samples while Zap-70(high) were completely resistant to TRAIL. The gene expression profiling was next analyzed in all B-CLL samples treated with either chlorambucil or recombinant TRAIL. While chlorambucil up-regulated the steady-state mRNA levels of known p53 target genes, such as PUMA, Fas/CD95 and MDM2 in all B-CLL samples examined, it significantly down-regulated survivin in Zap-70(low) but not in Zap-70(high). On the other hand, recombinant TRAIL up-regulated the expression of several cytokines (IL-1beta, IL-1alpha, IL-8), which have been involved in promoting B-CLL cell survival. In particular, TRAIL selectively up-regulated IL-1beta in Zap-70(low) B-CLL samples, while it markedly and selectively up-regulated its own mRNA and that of cyclooxigenase-2 (COX-2) in Zap-70(high). Taken together, our findings suggest that a significant expression of Zap-70 modulate the response of B-CLL to TRAIL, which might represents an initial step in the pathogenesis of B-CLL.


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacology , ZAP-70 Protein-Tyrosine Kinase/metabolism , Antineoplastic Agents, Alkylating/pharmacology , Chlorambucil/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , In Vitro Techniques , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Recombinant Proteins/pharmacology
10.
Blood ; 107(10): 4122-9, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16439677

ABSTRACT

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However, it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3, a small molecule inhibitor of the p53/MDM2 interaction, is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells, but not on normal CD19(+) B lymphocytes, peripheral-blood mononuclear cells, or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined, only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway, nutlin-3 up-regulated the steady-state mRNA levels of PCNA, CDKN1A/p21, GDF15, TNFRSF10B/TRAIL-R2, TP53I3/PIG3, and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover, nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL.


Subject(s)
Imidazoles/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Piperazines/pharmacology , Tumor Suppressor Protein p53/blood , Aged , Aged, 80 and over , Apoptosis/drug effects , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Neoplasm Staging , Reference Values
11.
J Cell Physiol ; 207(1): 158-64, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16270354

ABSTRACT

B chronic lymphocytic leukemia (B-CLL) cells express several members of the tumor necrosis factor (TNF) family, such as CD40L, CD30L, and TRAIL. By using the cDNA microarray technology, B-CLL samples were found to overexpress receptor activator of nuclear factor kB (NF-kB) ligand (RANKL), as compared to normal CD19(+) B cells. These findings were validated at the protein level by Western blot and flow cytometry analyses. Moreover, unlike primary normal B cells, leukemic B-CLL cells showed surface expression of RANK, the cognate transmembrane receptor of RANKL. When added in vitro to B-CLL cultures, either alone or in association with chlorambucil or fludarabine, recombinant RANKL did not significantly modulate cell viability, and it minimally affected the IL-8 expression/release. On the other hand, treatment with RANK-Fc chimera potently upregulated the release of IL-8 in the B-CLL culture supernatants, suggesting involvement of reverse signaling through transmembrane RANKL in IL-8 induction. In turn, exposure of B-CLL cells to recombinant IL-8 significantly decreased spontaneous apoptosis as well as chlorambucil- and fludarabine-mediated cytoxicity in B-CLL cells. Since IL-8 has been implicated in progression of B-CLL disease, our findings suggest that, by upregulating IL-8, the RANKL/RANK system may contribute to the pathogenesis of B-CLL.


Subject(s)
Carrier Proteins/metabolism , Glycoproteins/metabolism , Interleukin-8/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Survival/drug effects , Chlorambucil/pharmacology , Flow Cytometry , Gene Expression/genetics , Glycoproteins/genetics , Humans , Immunoglobulin G/genetics , Interleukin-1/pharmacology , Interleukin-8/genetics , Interleukin-8/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Oligonucleotide Array Sequence Analysis , Osteoprotegerin , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Up-Regulation/genetics , Vidarabine/analogs & derivatives , Vidarabine/pharmacology
12.
Am J Pathol ; 167(6): 1599-607, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16314473

ABSTRACT

Evidence suggests that cyclooxygenase-2 (COX-2) increases tumorigenic potential by promoting resistance to apoptosis. Because B chronic lymphoid leukemia (B-CLL) cells exhibit a defective apoptotic response, we analyzed CD19(+) B lymphocytes purified from the peripheral blood of B-CLL patients. Microarray analysis showed a variable (up to 38-fold) increase in the steady-state mRNA levels of COX-2 in B-CLL lymphocytes compared with normal CD19(+) B lymphocytes. The up-regulation of COX-2 in B-CLL cells was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. Moreover, immunohistochemical analysis of B-CLL bone marrow infiltrates confirmed clear expression of COX-2 in leukemic cells. Ex vivo treatment with the COX-2 inhibitor NS-398 significantly decreased the survival of leukemic cells by increasing the rate of spontaneous apoptosis in 13 of 16 B-CLL samples examined, but it did not affect the survival of normal lymphocytes. Pretreatment with NS-398 significantly potentiated the cytotoxicity induced by chlorambucil in 8 of 16 B-CLL samples examined. Moreover, although recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Apo2L showed little cytotoxic effect in most B-CLL samples examined, pretreatment with NS-398 sensitized 8 of 16 B-CLL samples to TRAIL-induced apoptosis. Taken together, our data indicate that COX-2 overexpression likely represents an additional mechanism of resistance to apoptosis in B-CLL and that pharmacological suppression of COX-2 might enhance chemotherapy-mediated apoptosis.


Subject(s)
Cyclooxygenase 2/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Membrane Proteins/genetics , Aged , Aged, 80 and over , B-Lymphocytes/enzymology , Cell Division , Female , Flow Cytometry , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lymphocytes/enzymology , Lymphocytes/pathology , Male , Middle Aged , Neoplasm Staging
13.
J Cell Physiol ; 205(2): 246-52, 2005 Nov.
Article in English | MEDLINE | ID: mdl-15887227

ABSTRACT

Analysis of peripheral blood (>85% CD19+/CD5+ B) lymphocytes, obtained from 44 patients affected by B chronic lymphoid leukemia (B-CLL), showed that surface TNF-related apoptosis inducing ligand (TRAIL) was expressed in all samples and at higher levels with respect to unfractionated lymphocytes and purified CD19+ B cells, obtained from 15 normal blood donors. Of note, in a subset of B-CLL samples, the addition to B-CLL cultures of a TRAIL-R1-Fc chimera, which binds at high affinity to surface TRAIL, significantly decreased the percentage of viable cells with respect to untreated control B-CLL cells, suggesting that surface TRAIL may play an unexpected role in promoting B-CLL cell survival. In spite of the majority of B-CLL lymphocytes expressed variable surface levels of "death receptors" TRAIL-R1 and TRAIL-R2, the addition in culture of recombinant TRAIL increased (>20% vs. controls) the degree of spontaneous apoptosis in only 11/44 of the B-CLL samples, had no effect in 19/44, while it significantly increased leukemic cell survival in 14/44. Taken together, these findings suggest that an aberrant expression of TRAIL might contribute to the pathogenesis of B-CLL by promoting the survival in a subset of B-CLL cells.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Membrane Glycoproteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal/metabolism , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/pharmacology , Blotting, Western , Case-Control Studies , Cell Survival/drug effects , Cells, Cultured , Female , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukocytes, Mononuclear/metabolism , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Middle Aged , RNA, Messenger/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor Decoy Receptors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/pharmacology
14.
Blood ; 105(9): 3413-9, 2005 May 01.
Article in English | MEDLINE | ID: mdl-15644410

ABSTRACT

Exposure of endothelial cells to recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced a modest (2-fold) increase of HL-60 cell adhesion as compared to TNF-alpha (40-fold) or interleukin 1beta (IL-1beta; 20-fold). However, pretreatment of endothelial cultures with TRAIL determined a significant reduction of the proadhesive activity induced by both TNF-alpha and IL-1beta. Unexpectedly, the antiadhesive activity of TRAIL was not due to interference with the nuclear factor kappaB (NF-kappaB)-mediated up-regulation of surface intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin adhesion molecules in response to inflammatory cytokines. In searching for the molecular mechanism underlying this biologic activity of TRAIL, a cDNA microarray analysis was performed. TRAIL pretreatment variably down-modulated the mRNA steady-state levels of several TNF-alpha-induced chemokines, and, in particular, it abrogated the TNF-alpha-mediated up-regulation of CCL8 and CXCL10. Of note, the addition of optimal concentrations of recombinant CCL8 plus CXCL10 to endothelial cultures completely restored the proadhesive activity of TNF-alpha. Moreover, experiments performed with agonistic anti-TRAIL receptor antibodies demonstrated that both TRAIL-R1 and TRAIL-R2 contributed, although at different levels, to TRAIL-induced chemokine modulation. Taken together, our data suggest that TRAIL might play an important role in modulating leukocyte/endothelial cell adhesion by selectively down-regulating CCL8 and CXCL10 chemokines.


Subject(s)
Chemokines, CXC/genetics , Cytokines/pharmacology , Down-Regulation/drug effects , Endothelium, Vascular/cytology , Membrane Glycoproteins/pharmacology , Monocyte Chemoattractant Proteins/genetics , Tumor Necrosis Factor-alpha/pharmacology , Apoptosis Regulatory Proteins , Cell Adhesion/drug effects , Chemokine CCL8 , Chemokine CXCL10 , Chemokines, CXC/metabolism , Coculture Techniques , Drug Antagonism , Gene Expression Regulation/drug effects , HL-60 Cells , Humans , Inflammation , Leukocytes/cytology , Monocyte Chemoattractant Proteins/metabolism , RNA, Messenger/analysis , RNA, Messenger/drug effects , TNF-Related Apoptosis-Inducing Ligand , Umbilical Veins/cytology
15.
Exp Hematol ; 32(10): 925-34, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15504548

ABSTRACT

OBJECTIVE: The present study was aimed at characterizing the expression and activity of cyclooxygenase (COX) isoenzymes in erythropoiesis. METHODS: The expression and activity of cyclooxygenase (COX) and prostaglandin (PG) synthases were investigated in: 1) erythroblasts developed in culture from human CD34(+) hematopoietic progenitors, 2) erythroblasts in bone marrow specimens, and 3) peripheral erythrocytes isolated from healthy donors and from patients with a high regeneration rate of erythrocytes. RESULTS: While COX-1 protein was observed at each stage of erythroblast development, COX-2 protein was induced at later stages through a p38/MAPK-dependent pathway. Both COX isoforms were also observed in mature erythroblasts of the bone marrow. Erythroblasts developed in culture synthesized significantly more PGE(2) than TXB(2) and indomethacin delayed erythroid maturation. COX-1 and COX-2 were also observed in erythrocytes by immunostainings, although COX expression was confined to a fraction of circulating erythrocytes. Peripheral erythrocytes synthesized low but detectable amounts of PGE(2) and TXB(2). Similarly to erythroblast progenitors, PGE(2) was the prevalent prostanoid released by erythrocytes. This biosynthetic capacity was significantly increased in erythrocytes from patients with accelerated erythropoiesis as compared to controls. CONCLUSIONS: Both COX isoforms are present and enzymatically active during human erythropoiesis, although with different kinetics, and COX-derived prostanoids may play a role in erythroid maturation. Furthermore, peripheral erythrocytes retain in part the capacity of expressing COX and synthesizing prostanoids, which may contribute to the hemostatic/thrombotic response to vascular injury in different diseases, including congenital hemolytic disorders.


Subject(s)
Erythropoiesis , Gene Expression Regulation , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Adult , Bone and Bones/cytology , Cells, Cultured , Cyclooxygenase 1 , Cyclooxygenase 2 , Dinoprostone/biosynthesis , Erythroblasts/cytology , Erythroblasts/enzymology , Erythroblasts/metabolism , Erythrocytes/cytology , Erythrocytes/enzymology , Erythrocytes/metabolism , Female , Fetal Blood , Humans , Isoenzymes , Kinetics , Male , Membrane Proteins , Middle Aged , Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandins/biosynthesis , RNA, Messenger/analysis , Thromboxane B2/biosynthesis
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